Study design and Setting This was a single-center, investigator-blinded, cross-sectional study. Work was conducted at the Department of Periodontology in Istanbul, Turkey, between November 2018 and July 2019. The study procedure was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the university (Protocol code: 10840098-604.01.01-E.47596\&10840098-604.01.01-E.4759; date of approval: 30 October 2018). Data from six non-smoking participants with healthy periodontium from our previous studies were used in this study as well. After the study was verbally explained to eligible subjects, those willing to participate signed a written, informed consent form. Personal, identifiable information about the subjects was kept confidential. The study was reviewed following the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.
Participants Eighty systemically healthy subjects, both male and female, were enrolled in the study. Participants were recruited from patients who came to our department for periodontal examinations between November 2018 and July 2019. Diagnoses of clinically healthy periodontium and generalized periodontitis stages III-IV/grades B-C were performed according to the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions. Subjects were separated into four groups: smokers with generalized periodontitis (SGP; n = 20), non-smokers with generalized periodontitis (NGP; n = 20), smokers with clinically healthy periodontium (SHP; n = 20), and non-smokers with clinically healthy periodontium (NHP; n = 20). The following criteria needed to be met to be considered a periodontitis subject: the presence of generalized periodontitis, the presence of at least 20 teeth, the presence of a molar with an indication of gingivectomy, crown lengthening or extraction with a probing depth ≥ 5mm, clinical attachment loss ≥ 5 mm, and the presence of at least ten teeth with a probing depth ≥ 5 mm. The following criteria indicated a clinically healthy periodontium: a healthy and intact periodontium, no probing depth and bleeding on probing.
Clinical procedure All subjects answered a questionnaire that assessed age, gender, and educational level. A clinical evaluation of periodontal disease was conducted according to international standards. The CPPs, including plaque index, probing depth, bleeding on probing, and clinical attachment loss, were recorded from six sites per tooth from each subject using a periodontal probe by the same researcher. The sample collection and histopathologic evaluation procedures were performed as in our previous report. Briefly, smear samples were collected from the attached gingival mucosa of the upper premolar-molar area and the buccal mucosa of the matching cheek using two separate sterile cytobrushes. Samples were fixed on glass slides. Biopsy samples were taken from the vestibular area of the maxillary molars during tooth extraction or crown lengthening using a surgical blade and fixed in 10% formalin until analysis. During microscopic inspection, each subject rinsed their mouth before samples were obtained to reduce the possibility of artifacts through chromatin remainders.
Laboratory procedure To evaluate the exfoliated oral epithelial cells containing micronuclei counts (the primary outcome), the Feulgen reaction was performed on all smear samples. Cells were placed in a fixative for nuclear staining and analysis using Schiff's reagent. Micronuclei in the buccal and gingival smear samples were analyzed by observing them in oil immersion at 1000× magnification. One thousand cells were checked, and the count of micronuclei present was assessed. The occurrence of nuclear morphological changes, such as the presence of two nuclei within a cell, nuclei that appeared cinched with a Feulgen negative band, shrunken nuclei, condensed chromatin, nuclear disintegration involving the loss of nuclear integrity, and nuclear dissolution was estimated. A zigzag strategy was used to calculate the cells.
Histopathologic damage (the secondary outcome) was evaluated by focusing on inflammatory cell and vascular densities. Damage was assessed using a modified semi-quantitative histopathologic assessment method using a scale ranging from 0 to 3 (0: none, 1: mild, 2: moderate, and 3: intense). Tissue samples were fixed in paraffin wax, serially sectioned at a 4 µm thickness, stained with hematoxylin-eosin, and examined at 400× magnification. All smear and tissue samples were assayed concurrently and in the same laboratory by the same blinded investigator.
Bias The evaluation of each participant's systemic health status, dental history, and smoking habits was based on self-reported information regardless of official medical records. One researcher collected the data and samples from the participants while another researcher undertook the laboratory investigations. To avoid bias, five sample areas were randomly selected during the histologic examinations.
Study size The sample size was determined using G\*Power version 3.1.9.4, assuming an alpha significance level of 0.05, a beta value of 0.1, and 90% power. As there were four study groups and a need for 20 participants per group, eighty subjects were included in the study.
Statistical methods The Number Cruncher Statistical System 2007 software program was used for all statistical analyses. Descriptive statistics (mean, standard deviation, median) were used to evaluate the data. The one-way analysis of variance test was used to compare the means of three or more normally distributed parameters. The Bonferroni test was used to determine the significant differences in pairwise comparisons between the groups. The Pearson chi-square test was used to compare qualitative data. Significance was evaluated at the p\<0.01 and p\<0.05 levels.
